RESUMO
Acute myeloid leukemia (AML) is a complex malignancy with poor prognosis. Several genetic lesions can lead to the disease. One of these corresponds to the NUP98-HOXA9 (NA9) translocation that fuses sequences encoding the N-terminal part of NUP98 to those encoding the DNA-binding domain of HOXA9. Despite several studies, the mechanism underlying NA9 ability to induce leukemia is still unclear. To bridge this gap, we sought to functionally dissect NA9 activity using Drosophila. For this, we generated transgenic NA9 fly lines and expressed the oncoprotein during larval hematopoiesis. This markedly enhanced cell proliferation and tissue growth, but did not alter cell fate specification. Moreover, reminiscent to NA9 activity in mammals, strong cooperation was observed between NA9 and the MEIS homolog HTH. Genetic characterization of NA9-induced phenotypes suggested interference with PVR (Flt1-4 RTK homolog) signaling, which is similar to functional interactions observed in mammals between Flt3 and HOXA9 in leukemia. Finally, NA9 expression was also found to induce non-cell autonomous effects, raising the possibility that its leukemia-inducing activity also relies on this property. Together, our work suggests that NA9 ability to induce blood cell expansion is evolutionarily conserved. The amenability of NA9 activity to a genetically-tractable system should facilitate unraveling its molecular underpinnings.
Assuntos
Drosophila melanogaster/crescimento & desenvolvimento , Drosophila melanogaster/metabolismo , Hematopoese , Proteínas de Homeodomínio/metabolismo , Tecido Linfoide/crescimento & desenvolvimento , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Animais , Diferenciação Celular , Proliferação de Células , Proteínas de Drosophila/metabolismo , Hemócitos/patologia , Humanos , Hiperplasia , Tecido Linfoide/patologia , Mamíferos , Índice Mitótico , Fenótipo , Receptores Proteína Tirosina Quinases/metabolismo , Transdução de Sinais , Células-Tronco/citologiaRESUMO
Valosin-containing protein/p97 is an ATP-driven protein segregase that cooperates with distinct protein cofactors to control various aspects of cellular homeostasis. Mutations at the interface between the regulatory N-domain and the first of two ATPase domains (D1 and D2) deregulate the ATPase activity and cause a multisystem degenerative disorder, inclusion body myopathy associated with Paget disease of bone and frontotemporal dementia/amyotrophic lateral sclerosis. Intriguingly, the mutations affect only a subset of p97-mediated pathways correlating with unbalanced cofactor interactions and most prominently compromised binding of the ubiquitin regulatory X domain-containing protein 1 (UBXD1) cofactor during endolysosomal sorting of caveolin-1. However, how the mutations impinge on the p97-cofactor interplay is unclear so far. In cell-based endosomal localization studies, we identified a critical role of the N-terminal region of UBXD1 (UBXD1-N). Biophysical studies using NMR and CD spectroscopy revealed that UBXD1-N can be classified as intrinsically disordered. NMR titration experiments confirmed a valosin-containing protein/p97 interaction motif and identified a second binding site at helices 1 and 2 of UBXD1-N as binding interfaces for p97. In reverse titration experiments, we identified two distant epitopes on the p97 N-domain that include disease-associated residues and an additional interaction between UBXD1-N and the D1D2 barrel of p97 that was confirmed by fluorescence anisotropy. Functionally, binding of UBXD1-N to p97 led to a reduction of ATPase activity and partial protection from proteolysis. These findings indicate that UBXD1-N intercalates into the p97-ND1 interface, thereby modulating interdomain communication of p97 domains and its activity with relevance for disease pathogenesis. We propose that the polyvalent binding mode characterized for UBXD1-N is a more general principle that defines a subset of p97 cofactors.
Assuntos
Adenosina Trifosfatases/metabolismo , Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Proteínas Adaptadoras de Transporte Vesicular , Motivos de Aminoácidos , Proteínas Relacionadas à Autofagia , Sítios de Ligação , Proteínas de Transporte/química , Caveolina 1/metabolismo , Linhagem Celular , Dicroísmo Circular , Endossomos/metabolismo , Epitopos/química , Polarização de Fluorescência , Proteínas de Fluorescência Verde/metabolismo , Homeostase , Humanos , Lisossomos/metabolismo , Espectroscopia de Ressonância Magnética , Mutação , Proteínas Nucleares/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Ubiquitina/química , Proteína com ValosinaRESUMO
The balance between protein and lipid biosynthesis and their eventual degradation is a critical component of cellular health. Autophagy, the catabolic process by which cytoplasmic material becomes degraded in lysosomes, can be induced by various physiological stimuli to maintain cellular homeostasis. Autophagy was for a long time considered a non-selective bulk process, but recent data have shown that unwanted components such as aberrant protein aggregates, dysfunctional organelles and invading pathogens can be selectively eliminated by autophagy. Recently, also intracellular lipid droplets were described as specific autophagic cargo, indicating that autophagy plays a role in lipid metabolism and storage (Singh et al., 2009 [1]). Moreover, over the past several years, it has become increasingly evident that lipids and lipid-modifying enzymes play important roles in the autophagy process itself, both at the level of regulation of autophagy and as membrane constituents required for formation of autophagic vesicles. In this review, we will discuss the interplay between lipids and autophagy, as well as the role of lipid-binding proteins in autophagy. We also comment on the possible implications of this mutual interaction in the context of disease. This article is part of a Special Issue entitled Lipids and Vesicular Transport.
Assuntos
Metabolismo dos Lipídeos , Fagossomos/metabolismo , Transdução de Sinais , Animais , Autofagia , Citoplasma/metabolismo , Proteínas de Ligação a Ácido Graxo/deficiência , Proteínas de Ligação a Ácido Graxo/genética , Expressão Gênica , Humanos , Transtornos do Metabolismo dos Lipídeos/metabolismo , Lisossomos/metabolismoRESUMO
MLL-rearranged (MLL-r) leukemias are among the leukemic subtypes with poorest survival, and treatment options have barely improved over the last decades. Despite increasing molecular understanding of the mechanisms behind these hematopoietic malignancies, this knowledge has had poor translation into the clinic. Here, we report a Drosophila melanogaster model system to explore the pathways affected in MLL-r leukemia. We show that expression of the human leukemic oncogene MLL-AF4 in the Drosophila hematopoietic system resulted in increased levels of circulating hemocytes and an enlargement of the larval hematopoietic organ, the lymph gland. Strikingly, depletion of Drosophila orthologs of known interactors of MLL-AF4, such as DOT1L, rescued the leukemic phenotype. In agreement, treatment with small-molecule inhibitors of DOT1L also prevented the MLL-AF4-induced leukemia-like phenotype. Taken together, this model provides an in vivo system to unravel the genetic interactors involved in leukemogenesis and offers a system for improved biological understanding of MLL-r leukemia.
RESUMO
Cells in living organisms are dynamic compartments that continuously respond to changes in their environment to maintain physiological homeostasis. While basal autophagy exists in cells to aid in the regular turnover of intracellular material, autophagy is also a critical cellular response to stress, such as nutritional depletion. Conversely, the deregulation of autophagy is linked to several diseases, such as cancer, and hence, autophagy constitutes a potential therapeutic target. Image analysis to follow autophagy in cells, especially on high-content screens, has proven to be a bottleneck. Machine learning (ML) algorithms have recently emerged as crucial in analyzing images to efficiently extract information, thus contributing to a better understanding of the questions at hand. This paper presents CELLULAR, an open dataset consisting of images of cells expressing the autophagy reporter mRFP-EGFP-Atg8a with cell-specific segmentation masks. Each cell is annotated into either basal autophagy, activated autophagy, or unknown. Furthermore, we introduce some preliminary experiments using the dataset that can be used as a baseline for future research.
Assuntos
Autofagia , Autofagia/fisiologia , Humanos , AnimaisRESUMO
Glioblastoma (GBM), a very aggressive and incurable tumor, often results from constitutive activation of EGFR (epidermal growth factor receptor) and of phosphoinositide 3-kinase (PI3K). To understand the role of autophagy in the pathogenesis of glial tumors in vivo, we used an established Drosophila melanogaster model of glioma based on overexpression in larval glial cells of an active human EGFR and of the PI3K homolog Pi3K92E/Dp110. Interestingly, the resulting hyperplastic glia express high levels of key components of the lysosomal-autophagic compartment, including vacuolar-type H+-ATPase (V-ATPase) subunits and ref(2)P (refractory to Sigma P), the Drosophila homolog of SQSTM1/p62. However, cellular clearance of autophagic cargoes appears inhibited upstream of autophagosome formation. Remarkably, downregulation of subunits of V-ATPase, of Pdk1, or of the Tor (Target of rapamycin) complex 1 (TORC1) component raptor prevents overgrowth and normalize ref(2)P levels. In addition, downregulation of the V-ATPase subunit VhaPPA1-1 reduces Akt and Tor-dependent signaling and restores clearance. Consistent with evidence in flies, neurospheres from patients with high V-ATPase subunit expression show inhibition of autophagy. Altogether, our data suggest that autophagy is repressed during glial tumorigenesis and that V-ATPase and MTORC1 components acting at lysosomes could represent therapeutic targets against GBM.
Assuntos
Neoplasias , ATPases Vacuolares Próton-Translocadoras , Animais , Autofagia/genética , Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Humanos , Lisossomos/metabolismo , Neoplasias/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , ATPases Vacuolares Próton-Translocadoras/metabolismoRESUMO
Macroautophagy/autophagy is a membrane trafficking and intracellular degradation process involving the formation of double-membrane autophagosomes and their ultimate fusion with lysosomes. Much is yet to be learned about the regulation of this process, especially at the level of the membranes and lipids involved. We have recently found that the PX domain protein HS1BP3 (HCLS1 binding protein 3) is a negative regulator of autophagosome formation. HS1BP3 depletion increases the formation of LC3-positive autophagosomes both in human cells and zebrafish. HS1BP3 localizes to ATG16L1- and ATG9-positive autophagosome precursors deriving from recycling endosomes, which appear to fuse with LC3-positive phagophores. The HS1BP3 PX domain interacts with phosphatidic acid (PA) and 3'-phosphorylated phosphoinositides. When HS1BP3 is depleted, the total cellular PA content is upregulated stemming from increased activity of the PA-producing enzyme PLD (phospholipase D) and increased localization of PLD1 to ATG16L1-positive membranes. We propose that HS1BP3 negatively regulates autophagy by decreasing the PA content of the ATG16L1-positive autophagosome precursor membranes through inhibition of PLD1 activity and localization.
Assuntos
Autofagia/fisiologia , Endossomos/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Fagossomos/metabolismo , Fosfolipase D/metabolismo , Animais , Proteínas Relacionadas à Autofagia/metabolismo , HumanosRESUMO
A fundamental question is how autophagosome formation is regulated. Here we show that the PX domain protein HS1BP3 is a negative regulator of autophagosome formation. HS1BP3 depletion increased the formation of LC3-positive autophagosomes and degradation of cargo both in human cell culture and in zebrafish. HS1BP3 is localized to ATG16L1- and ATG9-positive autophagosome precursors and we show that HS1BP3 binds phosphatidic acid (PA) through its PX domain. Furthermore, we find the total PA content of cells to be significantly upregulated in the absence of HS1BP3, as a result of increased activity of the PA-producing enzyme phospholipase D (PLD) and increased localization of PLD1 to ATG16L1-positive membranes. We propose that HS1BP3 regulates autophagy by modulating the PA content of the ATG16L1-positive autophagosome precursor membranes through PLD1 activity and localization. Our findings provide key insights into how autophagosome formation is regulated by a novel negative-feedback mechanism on membrane lipids.
Assuntos
Autofagia/fisiologia , Proteínas do Tecido Nervoso/metabolismo , Ácidos Fosfatídicos/metabolismo , Animais , Animais Geneticamente Modificados , Autofagossomos/metabolismo , Proteínas Relacionadas à Autofagia/metabolismo , Linhagem Celular , Cortactina/metabolismo , Células HEK293 , Células HeLa , Humanos , Lipídeos de Membrana/metabolismo , Modelos Biológicos , Proteínas do Tecido Nervoso/química , Fosfolipase D/metabolismo , Domínios Proteicos , Peixe-Zebra , Proteínas de Peixe-Zebra/metabolismoRESUMO
The Ras/MAPK-signaling pathway plays pivotal roles during development of metazoans by controlling cell proliferation and cell differentiation elicited, in several instances, by receptor tyrosine kinases (RTKs). While the internal mechanism of RTK-driven Ras/MAPK signaling is well understood, far less is known regarding its interplay with other co-required signaling events involved in developmental decisions. In a genetic screen designed to identify new regulators of RTK/Ras/MAPK signaling during Drosophila eye development, we identified the small GTPase Rap1, PDZ-GEF, and Canoe as components contributing to Ras/MAPK-mediated R7 cell differentiation. Rap1 signaling has recently been found to participate in assembling cadherin-based adherens junctions in various fly epithelial tissues. Here, we show that Rap1 activity is required for the integrity of the apical domains of developing photoreceptor cells and that reduced Rap1 signaling hampers the apical accumulation of the Sevenless RTK in presumptive R7 cells. It thus appears that, in addition to its role in cell-cell adhesion, Rap1 signaling controls the partitioning of the epithelial cell membrane, which in turn influences signaling events that rely on apico-basal cell polarity.
Assuntos
Proteínas de Drosophila/metabolismo , Drosophila/genética , Proteínas do Olho/metabolismo , Olho/embriologia , Receptores Proteína Tirosina Quinases/metabolismo , Proteínas rap1 de Ligação ao GTP/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Junções Aderentes/genética , Junções Aderentes/metabolismo , Animais , Adesão Celular/genética , Diferenciação Celular/genética , Polaridade Celular/genética , Mapeamento Cromossômico , Drosophila/embriologia , Proteínas de Drosophila/genética , Células Epiteliais/metabolismo , Proteínas do Olho/genética , Regulação da Expressão Gênica no Desenvolvimento , Sistema de Sinalização das MAP Quinases , Organogênese/genética , Células Fotorreceptoras de Invertebrados/metabolismo , Receptores Proteína Tirosina Quinases/genética , Proteínas rap1 de Ligação ao GTP/genéticaRESUMO
The role of membrane remodeling and phosphoinositide-binding proteins in autophagy remains elusive. PX domain proteins bind phosphoinositides and participate in membrane remodeling and trafficking events and we therefore hypothesized that one or several PX domain proteins are involved in autophagy. Indeed, the PX-BAR protein SNX18 was identified as a positive regulator of autophagosome formation using an image-based siRNA screen. We show that SNX18 interacts with ATG16L1 and LC3, and functions downstream of ATG14 and the class III PtdIns3K complex in autophagosome formation. SNX18 facilitates recruitment of ATG16L1 to perinuclear recycling endosomes, and its overexpression leads to tubulation of ATG16L1- and LC3-positive membranes. We propose that SNX18 promotes LC3 lipidation and tubulation of recycling endosomes to provide membrane for phagophore expansion.
Assuntos
Autofagia/fisiologia , Endossomos/metabolismo , Fagossomos/metabolismo , Nexinas de Classificação/metabolismo , Animais , Autofagia/genética , Proteínas de Transporte/metabolismo , Membrana Celular/metabolismo , HumanosRESUMO
The membrane remodeling events required for autophagosome biogenesis are still poorly understood. Because PX domain proteins mediate membrane remodeling and trafficking, we conducted an imaging-based siRNA screen for autophagosome formation targeting human PX proteins. The PX-BAR protein SNX18 was identified as a positive regulator of autophagosome formation, and its Drosophila melanogaster homologue SH3PX1 was found to be required for efficient autophagosome formation in the larval fat body. We show that SNX18 is required for recruitment of Atg16L1-positive recycling endosomes to a perinuclear area and for delivery of Atg16L1- and LC3-positive membranes to autophagosome precursors. We identify a direct interaction of SNX18 with LC3 and show that the pro-autophagic activity of SNX18 depends on its membrane binding and tubulation capacity. We also show that the function of SNX18 in membrane tubulation and autophagy is negatively regulated by phosphorylation of S233. We conclude that SNX18 promotes autophagosome formation by virtue of its ability to remodel membranes and provide membrane to forming autophagosomes.
Assuntos
Autofagia , Membrana Celular/metabolismo , Fagossomos/metabolismo , Nexinas de Classificação/metabolismo , Animais , Proteínas Relacionadas à Autofagia , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Drosophila/genética , Drosophila/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Endossomos/metabolismo , Corpo Adiposo/metabolismo , Células HEK293 , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Larva/genética , Larva/metabolismo , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Fosforilação , Mapeamento de Interação de Proteínas , Transporte Proteico , Proteólise , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Nexinas de Classificação/genéticaRESUMO
Ubiquitinated protein aggregates are hallmarks of a range of human diseases, including neurodegenerative, liver and muscle disorders. These protein aggregates are typically positive for the autophagy receptor p62. Whereas the ubiquitin-proteasome system (UPS) degrades shortlived and misfolded ubiquitinated proteins that are small enough to enter the narrow pore of the barrel-shaped proteasome, the lysosomal pathway of autophagy can degrade larger structures including entire organelles or protein aggregates. This degradation requires autophagy receptors that link the cargo with the molecular machinery of autophagy and is enhanced by certain posttranslational modifications of the cargo. In this review we focus on how autophagy clears aggregate-prone proteins and the relevance of this process to protein aggregate associated diseases.
Assuntos
Autofagia/fisiologia , Complexos Multiproteicos/metabolismo , Proteínas Ubiquitinadas/metabolismo , Acetilação , Transporte Ativo do Núcleo Celular , Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Relacionadas à Autofagia , Desacetilase 6 de Histona , Histona Desacetilases/química , Histona Desacetilases/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Modelos Biológicos , Complexos Multiproteicos/química , Doenças Neurodegenerativas/etiologia , Doenças Neurodegenerativas/metabolismo , Doenças Neurodegenerativas/terapia , Fosforilação , Complexo de Endopeptidases do Proteassoma/metabolismo , Transporte Proteico , Proteínas/química , Proteínas/metabolismo , Proteína Sequestossoma-1 , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo , Proteínas Ubiquitinadas/química , Ubiquitinação , Resposta a Proteínas não DobradasRESUMO
Reduced levels of autophagy correlate with tumorigenesis, and several inducers of autophagy have been found to be tumor suppressors. One such autophagic inducer is the Beclin 1 binding protein UVRAG, a positive regulator of the class III PI3K/Vps34 complex. UVRAG has been implicated in the formation and maturation of autophagosomes, as well as in endocytic trafficking and suppression of proliferation and in vivo tumorigenicity. In this study we show that approximately one-third of a large series of colon carcinomas with microsatellite instability (MSI) (n = 102) carry a monoallelic UVRAG mutation, leading to expression of a truncated protein, indicating that this event is involved in tumorigenesis. In order to investigate whether the high incidence of UVRAG mutation in MSI colorectal carcinomas is associated with dysfunctional autophagy we analyzed autophagy levels in several colon cancer cell lines that express wild-type or mutant UVRAG protein. No reduction in autophagy was detected in cell lines expressing mutant UVRAG. Consistent with this, depletion of UVRAG in HEK cells stably expressing GFP-LC3 did not inhibit autophagy, but did decrease epidermal growth factor receptor (EGFR) degradation. Overall our results show that there is no correlation between the presence of the monoallelic UVRAG mutation and inhibition of autophagy. Thus, our data indicate that mechanisms other than autophagy contribute to the tumorigenicity of microsatellite unstable colon carcinomas with monoallelic UVRAG mutation.